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* Institute of Clinical and Molecular Virology and
Department of Dermatology, University Hospital of Erlangen, Germany;
Institute of Medical Microbiology, University of Regensburg, Germany; and
Institute of Virology and Immunology, University of Wuerzburg, Germany
The main source for endogenous peptides presented by the MHC class I (MHC-I) pathway are de novo-synthesized proteins which are degraded via the ubiquitin proteasome pathway. Different MHC-I Ag pools can be distinguished: first, short-lived defective ribosomal products, which are degraded in concert with or shortly after their synthesis, and, second, functional proteins that enter the standard protein life cycle. To compare the contribution of these two Ag sources to the generation of MHC-I-presented peptides, we established murine cell lines which express as a model Ag the HIV-1 Gag polyprotein fused to ubiquitin (Ub) carrying the epitope SIINFEKL (SL). Gag was expressed either in its wild-type form (UbMGagSL) or as a variant UbRGagSL harboring an N-end rule degron signal. Although UbRGagSL displayed wild-type protein stability, its inherent defective ribosomal products rate observed after proteasome shutdown was increased concomitant with enhanced presentation of the SL epitope. In addition, UbRGagSL induces enhanced T cell stimulation of SL-specific B3Z hybridoma cells as measured in vitro and of adoptively transferred TCR-transgenic OT-1 T cells in vivo. Furthermore, an elevated frequency of SL-specific T cells was detected by IFN-
ELISPOT after immunization of naive C57BL/6 mice with UbRGagSL/EL4 cells. These results further underline the role of the defective ribosomal product pathway in adaptive immunity.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by a grant from the Bavarian Research Network for Infectogenomics, BioMedTec Internationational Graduate School of Science, "Lead Structures of Cell Functions," funded by the State of Bavaria, Germany, by Grant IE-S08T06 from the German Human Genome Research Project, and by Grant SFB 643-A1 from the German Research Council.
2 Current address: Institute of Clinical Microbiology, Immunology, and Hygiene, University Hospital of Erlangen, Erlangen, Germany.
3 Address correspondence and reprint requests to Dr. Ulrich Schubert, Institute of Clinical and Molecular Virology, University Hospital of Erlangen, Schlossgarten 4, 91054 Erlangen, Germany. E-mail address: ulrich.schubert{at}viro.med.uni-erlangen.de
4 Abbreviations used in this paper: MHC-I, MHC class I; ER, endoplasmic reticulum; DRiP, defective ribosomal product; UPS, ubiquitin proteasome system; Ub, ubiquitin; UFD, Ub fusion degradation; rVV, recombinant vaccinia virus; IAV, influenza virus A; NP, nucleoprotein; β-Gal, β-galactosidase; SL, SIINFEKL; Doc, deoxycholate; LN, lymph node; MFI, mean fluorescence intensity; LC, lactacystin; wt, wild type; DC, dendritic cell; zLLL, carbobenzoxyl-leucine-leucine-leucinal; CA, capsid.
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